Analgesics that act through the delta opioid receptor have low addictive potential but similarly to classical opiates, display tolerance after long-term treatment. Chronic opioid receptor stimulation leads to a compensatory increase in adenylyl cyclase (AC) activity, called AC superactivation. We have demonstrated that an AC isoenzyme (AC VI) is phosphorylated upon chronic 5 opioid agonist (SNC 80) treatment in CHO cells transfected with the human delta opioid receptor (hDOR/CHO). We hypothesize that phosphorylation of AC VI isinvolved in adenylyl cyclase superactivation, and that superactivation is involved in tolerance to chronic delta opioid agonists. In preliminary experiments we found that alpha-transducin, a putative scavenger of G protein beta-gamma-subunits, attenuated both chronic SNC 80 mediated phosphorylation of AC VI and AC superactivation. In this proposal we will use other, independent methods, to study the involvement of G protein beta-gamma-subunitsin cellular responses to acute- and chronic SNC 80 treatment. Attenuation of AC superactivation and AC VI phosphorylation by these methods will confirm the role of G protein beta-gamma-subunits in chronic SNC 80-mediated respolises. Subsequently we will show that free beta-gamma-subunits, released upon chronic SNC 80 treatment, regulate the activity of second messenger regulated protein kinases and Raf-1 protein kinase in hDOR/CHO cells. Finally, we will demonstrate that depletion of the protein kinase responsible for phosphorylation of AC VI in hDOR/CHO cells also attenuates chronic delta opioid agonist mediated AC superactivation. Better understanding of the molecular mechanisms of drug tolerance at the human delta opioid receptor should aid in the development of longer acting analgesics with fewer side effects.